DESCRIPTION: Hemophilia B is the X-linked bleeding disorder caused by absence of functional coagulation factor IX (F.IX). Pre-clinical studies in animal models have shown that gene transfer mediated by an adeno-associated viral (AAV) vector results in sustained expression of F.IX and partial correction of the coagulation deficiency. A Phase I clinical trial has been carried out based on intramuscular administration of vector to patients with severe hemophilia B, and a Phase I trial for liver-directed gene transfer (by infusion of the vector into the hepatic circulation) is now approved. Currently, the most serious complication of treatment for hemophilia by protein-based therapy is the formation of inhibitory antibodies against the coagulation factor. Using murine and canine models, we have demonstrated sustained F.IX expression with the muscle-directed approach in the context of a F.IX missense mutation, while expression in the context of a F.IX gene deletion/null mutation was limited by inhibitor formation. However, in animals of the same strain, sustained expression without inhibitor formation has been accomplished using liver-directed gene therapy. Thus, the immunological outcome of gene transfer is dependent on the combination of vector and target tissue. We found that AAV-mediated gene transfer to the liver can induce immunological unresponsiveness to F.IX, which may be explained by either a tolerance or a suppression mechanism. Anti-F.IX formation is dependent on CD4+ T helper cells. Therefore, we are proposing a gene transfer model based on mice transgenic for an ovalbumin CD4 about-restricted T cell receptor in order to define the events leading to antigen-specific immunity or unresponsiveness after AAV-mediated gene transfer of a secreted protein. We will investigate potential mechanisms of tolerance induction (clonal deletion, T cell anergy) or suppression/immune deviation (e.g. by activation of regulatory cells) in hepatic gene transfer as opposed to T cell priming associated with a neutralizing antibody response in lymph nodes of injected muscle. For both the ovalbumin and the F.IX system, we will perform adoptive lymphocyte transfer experiments to distinguish tolerance and suppression mechanisms in liver-directed gene transfer. In recently generated transgenic mice expressing liver-derived variants of human F.IX, the risk of inhibitor formation in liver-directed gene therapy can be directly compared to other treatment modalities after mice have been crossed with hemophilia B mice on the appropriate genetic background. Finally, the risk of inhibitor formation may be further reduced by a combination of liver-directed gene transfer and transient immune modulation. Taken together, these studies will provide a detailed analysis of transgene product-specific T cell responses following AAV-mediated hepatic gene transfer.